Anti-bag3 antibodies for therapeutic use

ABSTRACT

The present invention relates to the use of BAGS antibodies as a medicament, in particular for use in the treatment of pancreatic tumours or other pathologies of an immune, inflammatory, neoplastic and/or degenerative nature.

The present invention relates to the use of Anti-BAG3 antibodies as amedicament, in particular for use in the treatment of pancreatic tumoursor other pathologies of an immune, inflammatory, neoplastic and/ordegenerative nature.

STATE OF THE ART

BAG3 protein is a 74 kDa cytoplasmic protein which belongs to the familyof co-chaperonins that interact with the ATPase domain of the proteinHSP70 (Heat Shock Protein) through the structural domain known as theBAG domain (amino acids 110-124). Furthermore, BAG3 protein contains aWW domain (Trp-Trp), a proline-rich region (PXXP), and two conservedmotifs IPV (Ile-Pro-Val), which can mediate binding to other proteins.Thanks to the nature of BAG3 protein as an adapter, attributable to thepresence of many functional domains, such protein can therefore interactwith different proteins.

In humans, bag3 gene expression is constitutive for a few kinds ofnormal cells, including myocytes, while mutations thereof are associatedwith diseases of the skeletal and cardiac muscles. Furthermore, BAG3protein is expressed in many types of primary tumours or tumour celllines (lymphoid or myeloid leukemias, neuroblastoma, pancreatic cancer,thyroid cancer, breast cancer and prostate cancer, melanoma,osteosarcoma, glioblastoma and tumours of the kidney, colon, and ovary).

In normal cell types, such as leukocytes, epithelial cells and glialcells and cells of the retina, bag3 gene expression can be induced bystressors, such as oxidants, high temperatures, lack of serum, heavymetals, HIV-1 infections, etc. These findings indicate that bag3 geneexpression regulation is an important component in the cellular responseto stress and is correlated with the presence of elements that respondto the transcription factor HSF1 (Heat Shock Transcription Factor),which is activated in various forms of cellular stress in bag3 genepromoter (Franceschelli S., et al. J Cell Physiol 215 (2008) 575-577).

Moreover, due to the presence of many protein-protein interactiondomains in the structure thereof, BAG3 protein influences cell survivalin different types of cells, interacting with different molecularpartners. (A. Rosati et al. Cell Death Dis. (2011) 2:e141). The firstmechanism reported in relation to BAG3 anti-apoptotic activity wasidentified in osteosarcoma and melanoma cells, where it was observedthat BAG3 protein modulates the activation of transcription factor NF-kBand cell survival (Ammirante M. et al., Proc Natl Acad Sci USA 107(2010) 7497-7502). A different molecular mechanism has been described inglioblastoma cells, where BAG3 protein cooperates in a positive way withHSP70 protein to maintain BAX protein in the cytosol and prevent thetranslocation thereof into the mitochondria (Festa M. et al., Am JPathol 178 (2011) 2504-25). Finally, in some tumours, BAG3 has beenshown to regulate proteins that modulate cell adhesion.

The presence of cytoplasmic BAG3 protein has also been described in manydifferent cellular systems and has been associated, not only withvarious tumours, but also in pathologies in general related to cellsurvival.

Furthermore, patent application n. WO2011/067377 describes soluble BAG3protein, secreted externally to the cell, as a biochemical marker inserum, which is highly specific for the diagnosis of certainpathological conditions, such as cardiac pathologies and pancreatictumour. In particular, it has been demonstrated that, in patientssuffering from pancreatic adenocarcinoma, the concentration of(extra-cellular) soluble BAG3 is generally greater than 10 ng/ml.

Furthermore, it has recently been reported that BAG3 protein isexpressed in 346/346 patients with pancreatic ductal adenocarcinoma(PDAC) and is released by the cells of the pancreatic tumour, but suchprotein is not expressed in either the surrounding non-neoplastictissues or in a normal pancreas; likewise, it has been reported that thelevels of BAG3 expression are related to patient survival. The resultsof the study demonstrate that the use of specific siRNA molecules forBAG3 mRNA can silence bag3 gene expression and induce cell death,confirming that BAG3 protein is an important survival factor forpancreatic tumour cells and that the down-regulation thereof, whencombined with gentamicin, may contribute to the eradication of the tumorcells (Rosati et al., Am J Pathol. 2012 November; 181 (5):1524-9).

As it is known, conventional chemotherapy treatments for tumourpathologies, as well as treatments of inflammatory and immune diseaseswith corticosteroids or NSAIDs (non-steroidal anti-inflammatory drugs)pose numerous drawbacks linked to side effects and are not, at present,definitive means of treating such pathologies.

There is therefore an evident need for a new and improved therapeutictreatment which has the advantage of being highly specific and havingfew or no side effects, as compared with the conventional, commonlyknown therapies used for the treatment of diseases of an inflammatory,immune, and neoplastic nature described in the present invention.

DESCRIPTION OF THE INVENTION

Surprisingly, it has been demonstrated, for the first time, that theinhibition of soluble (i.e. extra-cellular) BAG3 protein through the useof anti-BAG3 monoclonal antibodies, impairs development of pancreatictumour cells. Anti-BAG3 antibodies represent a new and improvedtherapeutic tool for the treatment of pancreatic tumours. Furthermore,it has also been found, surprisingly, that the aforesaid BAG3 protein isinvolved in the activation of macrophages.

Therefore, treatment with any of the anti-BAG3 antibodies described inpatent application n. WO03/055908 able to inhibit, specifically, theactivity of soluble BAG3 protein (i.e. extra-cellular) on macrophages,that are considered the target cells, proves particularly effective inthe treatment of those pathologies characterised by the activation ofmacrophages, such as neoplastic diseases and diseases of aninflammatory, immune, or degenerative nature. In fact, these specificantibodies for BAG3 can bind and block, in a highly selective andtargeted manner, pathological effects related to BAG3 protein whensecreted by cells.

In particular, the use of anti-BAG3 antibodies in this process has thesurprising advantage of being more specific for the selectedpathological states characterised by the over-expression and release ofBAG3 protein, and also less damaging in terms of side effects.

The term “soluble BAG3 protein” is understood as extra-cellular BAG3protein, i.e. the protein secreted externally to the cell.

One aim of the present invention is therefore the use of anti-BAG3antibodies as a medicament.

The antibodies useable in accordance with the present invention may beeither monoclonal or polyclonal antibodies, and are preferablymonoclonal antibodies.

Still more preferably, said monoclonal antibodies may be chosen from thefollowing: murinr antibodies, humanised antibodies, chimeric antibodies,recombinant antibodies, conjugated antibodies, scFv fragments (diabody,triabody and tetrabody), Fab fragments, and fragments F (ab′) 2.

The term “polyclonal antibody” refers to a mixture of antibodies whichare genetically different since produced by different plasma cells andwhich recognise a different epitope of the same antigen.

The term “monoclonal antibody” refers to a set of antibodies which areall identical since produced by cell lines from only one type of immunecell (i.e. a cell clone).

The term “humanized antibody” refers to an antibody of human origin,whose hypervariable region has been replaced by the homologous region ofnon-human monoclonal antibodies.

The term “chimeric antibody” refers to an antibody containing portionsderived from different antibodies.

The term “recombinant antibody” refers to an antibody obtained usingrecombinant DNA methods.

The term “conjugated antibody” refers to antibodies conjugated withdrugs, toxins, radioactive substances or other agents.

The term “scFv fragment” (single chain variable fragment) refers toimmunoglobulin fragments only capable of binding with the antigenconcerned. ScFv fragments can also be synthesised into dimers(diabodies), trimers (triabodies) and tetramers (tetrabodies) usingpeptide linkers.

The terms “Fab fragment” (antigen-binding fragment) and “Fab2 fragment”refer to immunoglobulin fragments consisting of a light chain linked tothe Fc fragment of the adjacent heavy chain, and such fragments aremonovalent antibodies. When the Fab portions are in pairs, the fragmentis called Fab2.

The term “hybridoma” refers to a cell producing monoclonal antibodies.

The monoclonal antibodies used in the examples were obtained byimmunising mice against four distinct BAG3 protein peptides using anymethod known to a person skilled in the art. Such peptides were chosenbecause they are BAG3 protein-specific and are not shared with any otherprotein, including BAG proteins.

The sequences of the four peptides are included in the BAG3 amino acidsequence (RefSeq: NP_(—)004272; Gene ID 9531) and are selected from thefollowing:

SEQ ID NO 1 DRDPLPPGWEIKIDPQ; (includes BAG3 protein amino acids 18-33); SEQ ID NO 2: SSPKSVATEERAAPS;(includes BAG3 protein amino acids 385-399); SEQ ID NO 3:DKGKKNAGNAEDPHT; (includes BAG3 protein amino acids 533-547);SEQ ID NO 4: NPSSMTDTPGNPAAP;(includes BAG3 protein amino acids 561-575).

Preferably, said antibodies may be obtained by means of the MultipleAntigene Peptide approach (MAP) (Keah H H et al., J Pept Res (1988); 51:2. Tam J P; Proc Natl acad Sci USA (1988), 85: 5409. Ota S, et al.,Cancer Res (2002), 62: 1471), using the following map constructs:

MAP-BAG3-1: nh2-DRDPLPPGWEIKIDPQ-MAP(which contains sequence SEQ ID NO: 1); MAP-BAG3-2:nh2-SSPKSVATEERAAPS-MAP (which contains sequence SEQ ID NO: 2);MAP-BAG3-3: nh2-DKGKKNAGNAEDPHT-MAP(which contains sequence SEQ ID NO: 3); MAP-BAG3-4:nh2-NPSSMTDTPGNPAAP-MAP (which contains sequence SEQ ID NO: 4);

According to a preferred embodiment of the present invention, saidpolyclonal anti-BAG3 antibodies are obtained by immunising the animalsagainst one of the four peptides of the sequences SEQ ID NO. 1-4 statedabove.

According to a preferred embodiment, the monoclonal anti-BAG3 antibodiesof the present invention are obtained by means of a standard procedure(Tassone P., et al., Tissue Antigens 51: 671 (1998)) using the fourMAP-BAG3 peptides described above and are produced by at least one ofthe nine mother clones chosen from the following: AC-1, AC-2, AC-3,AC-4, AC-5, AC-6, AC-7, AC-8, or AC-9 (described in WO03/055908), whichcontain specific hybridomas for each of the four MAP-BAG3 constructsused.

According to a further embodiment, the antibodies used are monoclonalanti-BAG3 antibodies obtained from at least one of the aforesaid motherclones, and preferably at least one chosen from the following: AC-1,AC-2, AC-3, AC-4, or AC-5. More preferably, said monoclonal antibodiesare obtained from at least one mother clone chosen from the following:AC-1, AC-2, and AC-3.

According to a further preferred embodiment, with the standard procedure(Ceran C, Cokol M, Cingöz S, Tasan I, Ozturk M, Yagci T. Novel anti-HER2monoclonal antibodies: synergy and antagonism with tumor necrosisfactor-α.BMC Cancer. 2012 Oct. 4; 12:450) and the immunisation of micewith a BAG3 recombinant protein, the monoclonal anti-BAG3 antibodiesenvisaged in the present invention are obtained from at least one of thefollowing clones: AC-rb1, AC-rb2, AC-rb3 and AC.rb4, and/or at least oneof the following subclones: AC-rb1a, AC-rb1b, AC-rb2a, AC-rb2b, AC-rb3a,AC-rb3b, AC-rb4a, and AC-rb4b. The monoclonal antibodies produced by allthese clones and subclones recognise the BAG3 recombinant protein in anELISA test. (Example 2).

Preferably, said monoclonal anti-BAG3 antibodies are those thatrecognise epitopes in the BAG3 protein amino acid sequence, whichinclude at least one of the following fragments: 18-33, 385-399, 533-547or 562-575.

A further aim of the present invention is the use of the aforesaidanti-BAG3 antibodies in the treatment of a particular pathological statewhich involves the activation of macrophages. Such pathological statecan be chosen from: neoplastic diseases, inflammatory diseases, immunediseases, and/or degenerative diseases.

Preferably, such neoplastic diseases may be either pancreatic tumour orbladder tumor, more preferably pancreatic tumour.

Preferably, said inflammatory diseases can be chosen from diseasesrelated to inflammation of the skin, nerves, bones, blood vessels, andconnective tissues, and more preferably, psoriasis, arthritis, neuritis,connectivitis.

Preferably, said immune diseases can be chosen from autoimmune diseasessuch as rheumatic diseases, connective tissue diseases, neuromusculardiseases, endocrine diseases, gastrointestinal diseases, haematologicdiseases, skin diseases, and vasculitis, and more preferably, rheumatoidarthritis, multiple sclerosis, connectivitis, lupus erythematosus,endometriosis, and ulcerative colitis. Preferably, said degenerativediseases can be chosen from neurodegenerative diseases and musculardegenerative diseases, and more preferably Alzheimer's disease,Parkinson's disease, and muscular dystrophy.

According to a more preferred embodiment of the invention, the anti-BAG3antibodies are used in the treatment of neoplastic diseases,inflammatory diseases, immune diseases, and degenerative diseases.

A further aim of the present invention is a pharmaceutical compositioncomprising the aforesaid anti-BAG3 antibody in association with at leastone pharmaceutically acceptable excipient.

A further object of the present invention is the use of said compositionas a medicament.

A preferred embodiment of the present invention is the use of thecomposition in the treatment of neoplastic diseases and diseases of aninflammatory, immune and/or degenerative nature.

The composition of the present invention can be formulated in a formsuitable for oral administration or in a form suitable for parenteral ortopical administration.

In a preferred embodiment of the present invention, said oral form canbe chosen from the following: tablets, capsules, solutions, suspensions,granules, and oily capsules.

In a further preferred embodiment of the present invention, said topicalform can be chosen from the following: cream, ointment, ointment,solution, suspension, eye drops, pessary, nebuliser solution, spray,powder, or gel.

In a further preferred embodiment of this invention, said parenteralform can be either an aqueous buffer solution or an oily suspension.

Said parenteral administration include administration by intramuscular,intravenous, intradermal, subcutaneous, intraperitoneal, intranodal, orintrasplenic means.

DESCRIPTION OF THE FIGURES

FIG. 1. Picture showing the reduction of tumour growth in mice treatedwith AC-rb2.

FIG. 2. Results of the reduction of tumour growth in situ.

FIG. 3. Results of reduction of tumour growth in the surrounding area.

EXAMPLES Example 1 Treatment of BALB/c Mice with Anti-BAG3 Antibody(AC-rb2)

Nude mice (nu/nu) female BALB/c 6 weeks (Charles Rivers Wilmington,Mass., USA) were caged (3 per cage) with food and water ad libitum andkept in 12 h light/dark cycles in standard, pathogen-free conditions.The research protocol was approved by the Ethics Committee in accordancewith Italian Ministry of Health official guidelines. After one week ofacclimatisation, the mice were subjected to inoculation of cancer cells.10 mice were used in total, each one individually identified. The entireexperiment was conducted in laminar flow hoods and all surgicalprocedures were performed in strict compliance with aseptic techniques.The mice were anesthetised with 100 mg/ml of ketamine HCl and 20 mg/mlof xylazine injected intraperitoneally; they were then subjected tolaparotomy and the tail of the pancreas was gently exteriorised.MIA-PaCa 2 RFP cells (2.5×10⁶) were resuspended in 40 microlitres of PBS1× in a 1 ml syringe; using a 25 G needle, the cells were injected intothe tail of the pancreas and the injection point was swabbed withsterile cotton. Once homeostasis was ascertained, the tail of thepancreas was repositioned in the abdomen and the wound closed. After twoweeks, the mice were randomised into two groups: the first received anintraperitoneal injection of 100 mg/kg of mouse IgG and the second 100mg/kg of a murine monoclonal anti-BAG3 antibody (AC-rb2). This treatmentwas performed twice a week in total and the mice were then anesthetisedagain to check the tumour area using Macro Fluo and LAS V3.7 software,by Leica Mycrosystems Ltd. In each imaging, the tumour mass wasdetermined by quantification of the fluorescent area.

Results

The results shown in FIGS. 1-3 demonstrate that the treatment with theAnti-BAG3 AC-rb2 antibody reduces the growth of the tumour mass by over75% in situ and over 45% considering the area surrounding the tumour,i.e. the spread of the tumour.

Similar results were obtained with the murine monoclonal antibodyAC-rb3.

Example 2 ELISA Test on the Antibodies Obtained from the SubclonesAC-rb1a, AC-rb1b, AC-rb2a, AC-rb2b, AC-rb3a, AC-rb3b, AC-rb4a andAC-rb4b

The NUNC Maxisorp 96-well microtitre plates were functionalised withBAG3 recombinant protein 1 μg/ml in PBS 1×pH 7 (50 μl/well) andincubated for 18 hours at 4° C. The plates were then washed twice with abuffer wash (PBS 1×+0.05% Tween-20), and then kept in place for one hourat room temperature with 0.5% fish gelatin in PBS 1× (150 μl/well).Subsequently, the plates were buffer-washed twice and the supernatantsof the eight subclones were diluted 1/10, 1/100, and 1/1000, in a 0.5%fish gelatin in PBS 1× and then incubated (50 μl/well) in triplicate andincubated at room temperature for 2 hours. The plates were thenbuffer-washed six times. To develop the signal, murine anti-IgGantibodies (H+L) (Sigma Aldrich) were used, diluted in the same buffer1/20,000, then added to the plate (50 μl/well) and incubated at 4° C.for 30 minutes. After incubation, the plates were washed six times, thendeveloped with TMB (50 μl/well) (eBioscience), and the reaction wasstopped with sulphuric acid 4.5 M (25 μl/well). The plates were thenanalysed in a spectrophotometer at a wavelength of 450 nm. The resultsare expressed

AC-rb1 AC-rb2 AC-rb3 AC-rb4 AC- AC- AC- AC- AC- AC- AC- AC- Dilutionrb1a rb1b rb2a rb2b rb3a rb3b rb4a rb4b 1/10 0.778 0.773 1.051 0.9290.827 1.141 1.051 0.929 1/100 0.51 0.512 0.759 0.738 0.429 0.422 0.7590.738 1/1000 0.302 0.31 0.25 0.232 0.276 0.207 0.25 0.232as O.D. (optical densitometry).

The results obtained demonstrate that all the antibodies obtained by thesubclones tested were able to recognise BAG3 protein.

1-16. (canceled)
 17. A method for treating neoplastic diseases,inflammatory diseases, immune diseases and/or degenerative diseases,comprising administering an anti-BAG3 antibody to a subject in needthereof, wherein BAG3 protein is soluble.
 18. The method according toclaim 17, wherein said neoplastic disease is pancreatic tumor.
 19. Themethod according to claim 17, wherein said neoplastic disease is bladdertumor.
 20. The method according to claim 17, wherein said anti-BAG3antibody is a polyclonal antibody.
 21. The method according to claim 17,wherein said anti-BAG3 antibody is a monoclonal antibody.
 22. The methodaccording to claim 21, wherein said monoclonal antibody is a murineantibody, a humanized antibody, a chimeric antibody, a recombinantantibody, a conjugated antibody, an scFv fragment, a Fab fragment or aF(ab′)2 fragment.
 23. The method according to claim 22, wherein saidscFv fragment is diabody, triabody or tetrabody.
 24. The methodaccording to claim 22, wherein said monoclonal antibody is produced byat least one of the nine mother clones selected from the groupconsisting of: AC-1, AC-2, AC-3, AC-4, AC-5, AC-6, AC-7, AC-8 and AC-9.25. The method according to claim 24, wherein said monoclonal antibodyis produced by at least one of the five mother clones selected from thegroup consisting of: AC-1, AC-2, AC-3, AC-4, and AC-5.
 26. The methodaccording to claim 22, wherein said monoclonal antibody is produced byat least one of the following clones: AC-rb1, AC-rb2, AC-rb3, or AC-rb4,and by at least one of the following subclones: AC-rb1a, AC-rb1b,AC-rb2a, AC-rb2b, AC-rb3a, AC-rb3b, AC-rb4a, or AC-rb4b.
 27. Apharmaceutical composition comprising at least one anti-BAG3 antibodyand at least one pharmaceutically acceptable excipient, wherein the BAG3protein is soluble.
 28. The pharmaceutical composition according toclaim 27, wherein said anti-BAG3 antibody is a monoclonal antibodyproduced by at least one of the nine mother clones selected from thegroup consisting of: AC-1, AC-2, AC-3, AC-4, AC-5, AC-6, AC-7, AC-8 andAC-9.
 29. The pharmaceutical composition according to claim 27, whereinsaid anti-BAG3 antibody is a monoclonal antibody produced by at leastone of the five mother clones selected from the group consisting of:AC-1, AC-2, AC-3, AC-4, and AC-5.
 30. The pharmaceutical compositionaccording to claim 27, wherein said composition is formulated in a formsuitable for oral administration, parenteral administration, or topicaladministration.
 31. The pharmaceutical composition according to claim30, wherein said form suitable for oral administration is selected fromthe group consisting of tablets, capsules, solutions, suspensions,granules, and oily capsules.
 32. The pharmaceutical compositionaccording to claim 30, wherein said parenteral administration isintramuscular, intravenous, intradermal, subcutaneous, intraperitoneal,intranodal, or intrasplenic administration.
 33. The pharmaceuticalcomposition according to claim 30, wherein said form suitable fortopical administration is cream, salve, ointment, or gel.
 34. Thepharmaceutical composition according to claim 30, wherein said formsuitable for parenteral administration is an aqueous buffer solution oran oily suspension.